Simple method and kit for DNA typing of HLA genes by high-throughput massively parallel sequencer

ABSTRACT

The present invention addresses the problem of providing a method and kit for the DNA profiling of HLA genes using a high-throughput massively parallel sequencer. The present invention pertains to a method for the DNA profiling of HLA genes, said method being characterized by including: (1) a step for preparing a primer set that anneals specifically to exon 4 and intron 1 and includes exon 2 and exon 3 of at least one target gene selected from the group consisting of HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1 and HLA-DPB1 in the base sequence of the human genome; (2) a step for amplifying a sample (DNA) by PCR using the primer set; (3) a step for determining the base sequence of the amplified PCR product; and (4) a step for carrying out a homology search against a database.

TECHNICAL FIELD

The present invention relates to a method and a kit for DNA typing ofHLA genes using a high throughput massive parallel sequencer.

BACKGROUND ART

The human leukocyte antigen (HLA) plays a central role in immunologicaldiscrimination between self and non-self. This discrimination isachieved when T cells recognize, via T cell receptors, HLA-peptidecomplexes presenting self- or non-self-derived peptides on HLAs. T cellsrecognize cells expressing, on the surface, HLA-peptide complexespresenting non-self (pathogenic microbes such as viruses and bacteria,or foreign antigens such as pollens)-derived peptides on self-HLAs, orcells expressing, on the surface, non-self HLA alleles that have enteredthe body through transplantation or transfusion, thereby causingactivation of immunocytes or destroy of the presenting cells.

Such activation of immunocytes or destroy of the presenting cells causesa rejection response or graft versus host disease (GVHD) in transfusion,medical transplantation including bone marrow transplantation, orregenerative medicine using iPS cells or ES cells. In patients receivingfrequent platelet transfusion, an antibody against a non-self HLA isproduced and brings about a significant reduction in the efficacy of theplatelet transfusion. In some cases of medication, a drug (and apeptide) bound with a particular HLA may be recognized as a foreignsubstance, causing a severe adverse drug reaction based mostly on anallergy response.

Accordingly, medical transplantation or regenerative medicine requiresmatching of HLAs between a patient and a donor. Transfusion of“HLA-compatible platelet” with HLA match is also necessary for platelettransfusion patients in which an anti-HLA antibody against a particularallele is produced. For adverse drug reactions, it is also important toexamine HLAs before medication when the drug to be administered isreportedly related to a particular HLA allele. In actuality, packageinserts of some drugs clearly states recommendation to examine HLAs.Peptide vaccine therapy of cancer also requires examining HLAs forpredicting whether or not the peptide vaccine can bind to patient'sHLAs.

As major HLAs, six types of antigens are known, namely, class Imolecules (HLA-A, HLA-B and HLA-C), which are expressed in almost allcells, and class II molecules (HLA-DR, HLA-DQ and HLA-DP), which areexpressed mainly in immune cells.

The HLA class I antigen consists of a highly polymorphic α chain and asubstantially non-polymorphic β2-microglobulin; whereas the HLA class IIantigen consists of a highly polymorphic β chain and a less polymorphicα chain. The α chains of class I molecules are encoded by HLA-A, HLA-Band HLA-C genes. The β chains of class II antigens are encoded byHLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1 and HLA-DPB1 genes,whereas the α chains are encoded by HLA-DRA1, HLA-DQA1 and HLA-DPA1genes. In a gene level, in HLA class I antigens, exon 2 and exon 3 of agene encoding an α chain are highly polymorphic; whereas, in HLA classII antigens, exon 2 of a gene encoding a β chain is highly polymorphic.

A gene region encoding an HLA is located on short arm of humanchromosome 6 at 6p21.3. A class I region (HLA-A, HLA-C, HLA-B, etc.), aclass III region and a class II region (HLA-DRA, HLA-DRB1, HLA-DQA1,HLA-DQB1, HLA-DPA1, HLA-DPB1, etc.) are arranged in this order from thetelomere side toward the centromere side. Many genes are encoded at anextremely high density and association of these genes with transfusion,transplantation and various diseases has been reported. In the class IIIregion, no HLA genes are present and genes of complement components andtumor necrosis factors (TNF), etc. are present.

In an HLA-DRB gene region encoding a β chain of an HLA-DR antigen, ithas been confirmed that 5 types of structural polymorphisms are present.In DR1 type and DR10 type, pseudogenes such as HLA-DRB6 and HLA-DRB9 inaddition to HLA-DRB1 are located on the same chromosome. In DR2 type, anHLA-DRB5 (DR51) gene and pseudogenes such as HLA-DRB6 and HLA-DRB9 inaddition to HLA-DRB1 are located on the same chromosome. In DR3, DR5 andDR6 types, an HLA-DRB3 (DR52) gene and pseudogenes such as HLA-DRB2 andHLA-DRB9 in addition to HLA-DRB1 are located on the same chromosome. InDR4, DR7 and DR9 types, an HLA-DRB4 (DR53) gene and pseudogenes such asHLA-DRB7, HLA-DRB8 and HLA-DRB9 in addition to HLA-DRB1 are located onthe same chromosome. In contrast to these, in DR8 type, no HLA-DRB genesexcept HLA-DRB1 are located on the same chromosome (see FIG. 1).

In the exon of each allele, a plurality of regions exhibitingpolymorphism are present. In many cases, a nucleotide sequence (aminoacid sequence) present in a certain polymorphic region is commonlypresent in a plurality of alleles. In short, each HLA allele isspecified by a plurality of polymorphic regions in combination. In anHLA class II antigen, not only a polymorphic region in the exon but alsoexon 2 or exon 3 having the same nucleotide sequence is sometimescommonly present in a plurality of alleles.

Since a highly polymorphic region is present in an HLA, the number oftypes of alleles is known to be extremely large and notation of them hasbeen defined: i.e., a first field (two-digit level) is fordiscrimination of serologic HLA types, a second field (4-digit level) isfor discrimination of alleles having an amino acid substitution in thesame serologic HLA type, a third field (6-digit level) is fordiscrimination of alleles having a base substitution not accompanying anamino acid mutation and a fourth field (8-digit level) is fordiscrimination of alleles having a base substitution in an intron, whichis out of the genetic region encoding an HLA molecule (see FIG. 2).

In various medical cases, examination of HLA alleles (DNA typing of HLAgenes) is important. However, a SBT (sequence-based typing) method or aLuminex method (PCR-sequence-specific oligonucleotide probes(SSOP)-Luminex method), which has heretofore been frequently used,cannot easily determine whether polymorphic regions are in acis-configuration (on the same chromosome) or in a trans-configuration(on different chromosomes), if a plurality of different polymorphicregions are present between alleles. Therefore, the alleles weresometimes unable to be accurately determined due to the occurrence ofso-called phase ambiguity.

Hence, we developed a method capable of eliminating phase ambiguity byusing a next generation sequencer (high throughput massive parallelsequencer). In this method, however, typing of fragmented DNAs in a poorstate of preservation was sometimes not easily performed because a longregion containing 3′UTR was amplified from a promoter region.Particularly, an HLA class II gene has long intron 1, which requiresamplifying a region about twice longer than that of a class I gene.Therefore, typing of DNA fragment samples was not easily performed.

RELATED ART DOCUMENT Patent Document

-   Patent Document 1: JP H11-216000 A

Non Patent Document

-   Non Patent Document 1: Lind C. et al., Human Immunology, Vol. 71,    Pages 1033-1042 (2010)-   Non Patent Document 2: Shiina T. et al., Tissue Antigens, Vol. 80,    Pages 305-316 (2012)

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method, a primer setand a kit for DNA typing of HLA genes using a high throughput massiveparallel sequencer, which attain typing even in the case of usingfragmented DNA samples in a poor state of preservation and are capableof simultaneously PCR amplifying HLA genes using a plurality of primersets under the same PCR conditions.

Means for Solving the Problems

The present inventors developed a system in which a region from exon 2to a portion of exon 4 of an HLA class II gene, which is highlypolymorphic so as to bring about a difference between alleles andencodes an extracellular domain recognizable by an antibody or a T cellreceptor, is amplified and subjected to DNA typing using a nextgeneration sequencer.

Specifically, the present inventors came up with the new idea of newlydesigning each of PCR primers capable of simultaneously amplifyingHLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 genes, which are HLA class IIgenes, and PCR primers capable of specifically amplifying HLA-DQB1 andHLA-DPB1 genes, setting preferable PCR conditions, and using a highthroughput massive parallel sequencing technique. As a result ofperforming diligent research based on this idea in order to solve theabove-described problems, the present inventors completed the presentinvention.

In other words, the present invention provides a method for DNA typingof an HLA gene, including the following steps:

(1) a step of preparing a set of primers which anneal specifically to anintron 1 region containing exon 2 and exon 3 and an exon 4 region,respectively, of at least one target gene selected from the groupconsisting of HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1 andHLA-DPB1 genes in a human genome sequence;(2) a step of PCR amplifying a test sample (DNA) using the set ofprimers;(3) a step of determining the nucleotide sequence of the PCR amplifiedproduct; and(4) a step of carrying out a homology search within a database.

In one embodiment the target gene is at least one gene selected from thegroup consisting of HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 genes andthe set of primers is oligonucleotides having nucleotide sequences asshown in SEQ ID NOs: 1 and 2, respectively. In an alternativeembodiment, the target gene is an HLA-DQB1 gene and the set of primersis oligonucleotides having nucleotide sequences as shown in any one ofSEQ ID NOs: 3 and 4, or both, and SEQ ID NO: 5, respectively. In afurther alternative embodiment, the target gene is an HLA-DPB1 gene andthe set of primers is oligonucleotides having nucleotide sequences asshown in SEQ ID NOs: 6 and 7, respectively.

In another aspect, the present invention relates to a primer set capableof simultaneously PCR amplifying a DNA region including exon 2 and exon3 of at least one gene selected from the group consisting of HLA-DRB1,HLA-DRB3, HLA-DRB4 and HLA-DRB5 genes. In one embodiment, the presentinvention provides a primer set for DNA typing of at least one geneselected from the group consisting of HLA-DRB1, HLA-DRB3, HLA-DRB4 andHLA-DRB5 genes, including oligonucleotides having nucleotide sequencesas shown in SEQ ID NOs: 1 and 2, respectively. In an alternativeembodiment, the present invention provides a primer set for DNA typingof an HLA-DQB1 gene, including oligonucleotides having nucleotidesequences as shown in any one of SEQ ID NOs: 3 and 4, or both, and SEQID NO: 5, respectively. In a further alternative embodiment, the presentinvention provides a primer set for DNA typing of an HLA-DPB1 gene,including oligonucleotides having nucleotide sequences as shown in SEQID NOs: 6 and 7, respectively.

Effects of the Invention

The method of the present invention, since it provides all nucleotidesequences required for DNA typing of an HLA gene from a single molecule,is an ultimate DNA typing method in which phase ambiguity due to unclearcis/trans positional relationship is eliminated. Owing to this, a regionfrom exon 2 to a portion of exon 4 of an HLA class II gene, which ishighly polymorphic and encodes an extracellular domain recognizable byan antibody or a T cell receptor can be amplified and DNA typing with anext generation sequencer is realized.

Furthermore, since an amplification region is shortened, DNA typing ofan HLA gene can be relatively easily performed even in the case of usingDNA samples in a poor state of preservation. In addition, the length ofthe region to be amplified is nearly equal to an already publishedamplification region of a class I gene. Use of the primer sets of thepresent invention enables a plurality of HLA genes to be simultaneouslyPCR amplified under the same PCR conditions and can therefore shortenthe time required for PCR. Moreover, the data size is also reduced andthe time required for data analysis is therefore also shortened.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A diagram showing an HLA-DR gene region, cited from“Transplantation/transfusion Examination”, supervised by HidetoshiInoko, Takehiko Sasazuki and Takeo Juuji, Kodan-sha Scientific, 2004,page 48.

FIG. 2 A diagram showing a classification of HLA alleles, cited from theIMGT-HLA database (www.ebi.ac.uk/ipd/imgt/hla/).

FIG. 3 (a) A diagram showing the relationship between the structure ofan HLA class I gene and the structure of an HLA class I molecule; and(b) A diagram showing the structure of a promoter region of an HLA classI gene, cited from “Transplantation/transfusion Examination”, supervisedby Hidetoshi Inoko, Takehiko Sasazuki and Takeo Juuji, Kodan-shaScientific, 2004, page 35.

FIG. 4 A schematic diagram showing the structure of each HLA gene.

FIG. 5 A diagram showing the lengths of PCR products estimated from thepositions of primers designed on an HLA class II gene and referencesequences.

FIG. 6 An agarose gel electrophoretic pattern obtained using newlydeveloped primers.

FIG. 7 An agarose gel electrophoretic pattern obtained by a multiplexPCR.

FIG. 8 An agarose gel electrophoretic pattern obtained by a multiplexPCR.

MODES FOR CARRYING OUT THE INVENTION

Now, the DNA typing method of the present invention will be morespecifically described step by step.

(1) Step of Preparing a Primer Set

In the DNA typing method of the present invention, first, a set ofprimers which anneal specifically to intron 1 including exon 2 and exon3 and exon 4, respectively, of at least one target gene selected fromthe group consisting of HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1and HLA-DPB1 genes in a human genome sequence and can anneal under thesame conditions is prepared.

The genome sequence of human chromosome 6 (6p21.3) in which an HLA geneis present has been already elucidated and association between the genestructure and the structure of an expression product (HLA antigen) hasbeen known (see FIGS. 3 and 4).

In the present invention, a set of primers which can comprehensively PCRamplify regions including exon 2 and exon 3 of classic class II antigens(HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1 and HLA-DPB1) isprepared (see FIG. 5), and PCR products obtained by PCR amplificationusing the set of primers are subjected to high throughput sequencing(described later). Therefore, uncertainty such as phase ambiguity can beeliminated and the presence or absence of a null allele can beaccurately detected.

In Table 1, SEQ ID NOs: 1 and 2 represent a set of PCR primersspecifically amplifying an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4gene, and an HLA-DRB5 gene, which are β chains of MHC class II. Theseprimers of the set are nucleotide sequences located at positions, whichcorrespond to the upstream and downstream of exon 2 and exon 3 of eachof an HLA-DRB1 gene and an HLA-DRB5 gene in a human genome sequence(Reference sequence: hg19), an HLA-DRB1 gene and an HLA-DRB3 gene in ahuman genome sequence (Reference sequence: 6_cox_hap2) and an HLA-DRB1gene and an HLA-DRB4 gene in a human genome sequence (Referencesequence: mann_hap4), and sandwich these exons.

SEQ ID NO: 1 has a nucleotide sequence corresponding to the 32552643rdposition to the 32552667th position and the 32490446th position to the32490470th position in a human genome sequence (Reference sequence:hg19), the 4003862nd position to the 4003886th position and the3939870th position to the 3939894th position in a human genome sequence(Reference sequence: 6_cox_hap2) and the 4000197th position to the4000221st position and the 3851785th position to the 3851809th positionin a human genome sequence (Reference sequence: mann_hap4).

SEQ ID NO: 2 has a complementary nucleotide sequence to a nucleotidesequence corresponding to the 32548609th position to the 32548631stposition and the 32486419th position to the 32486441st position in ahuman genome sequence (Reference sequence: hg19), the 3999861st positionto the 3999883rd position and the 3935840th position to the 3935862ndposition in a human genome sequence (Reference sequence: 6_cox_hap2) andthe 3996203rd position to the 3996225th position and the 3847267thposition to the 3847289th position in a human genome sequence (Referencesequence: mann_hap4).

The length of a PCR product obtained by using this primer set isestimated from the reference sequence as about 4,000 to about 4,500bases (bp).

In Table 1, SEQ ID NOs: 3 to 5 represent a set of PCR primersspecifically amplifying an HLA-DQB1 gene, which is a β chain of MHCclass II. These primers of the set are nucleotide sequences located atpositions, which correspond to the upstream and downstream of exon 2 andexon 3 of an HLA-DQB1 gene and sandwich these exons, in a human genomesequence (Reference sequence: hg19).

SEQ ID NO: 3 has a nucleotide sequence corresponding to the 32633103rdposition to the 32633128th position in a human genome sequence(Reference sequence: hg19).

SEQ ID NO: 4 has a nucleotide sequence corresponding to the 32633103rdposition to the 32633127th position in a human genome sequence(Reference sequence: hg19).

SEQ ID NO: 5 has a complementary nucleotide sequence to a nucleotidesequence corresponding to the 32629214th position to the 32629237thposition in a human genome sequence (Reference sequence: hg19).

The length of a PCR product obtained by using this primer set isestimated from the reference sequence as about 3,900 bases (bp).

In Table 1, SEQ ID NOs: 6 and 7 represent a set of PCR primersspecifically amplifying an HLA-DPB1 gene, which is a β chain of MHCclass II. These primers of the set are nucleotide sequences located atpositions, which correspond to the upstream and downstream of exon 2 andexon 3 of an HLA-DPB1 gene and sandwich these exons, in a human genomesequence (Reference sequence: hg19).

SEQ ID NO: 6 has a nucleotide sequence corresponding to the 33048187thposition to the 33048207th position in a human genome sequence(Reference sequence: hg19).

SEQ ID NO: 7 has a complementary nucleotide sequence to a nucleotidesequence corresponding to the 33053563rd position to the 33053591stposition in a human genome sequence (Reference sequence: hg19).

The length of a PCR product obtained by using this primer set isestimated from the reference sequence as about 5,400 bases (bp).

These primers can be prepared by a method routinely used in this field.Furthermore, the sets of primers described in Table 1 are the mostpreferable examples. In the method of the present invention, any set ofprimers can be used as long as the set of primers is a set of a senseprimer and an anti-sense primer capable of annealing to the positions,which correspond to the upstream and downstream of exon 2 and exon 3 ofeach HLA gene and sandwich these exons.

Further, in the present specification, even if primers correspond to thesame region within the reference sequence, a separate sequence ID numberis assigned to each primer as long as they differ in the nucleotides.The difference in the nucleotide is due to a polymorphism.

TABLE 1 Length Estimated length of HLA of SEQ PCR product based class IIprimer Primer sequence ID on the reference gene Name of primer (mer)(5′-3′) NO sequence (bp) HLA-DRB1 DRB1-short-F201 25 TTCACTGCTCTTWAAGCTC1 4,059 (DRB1) HLA-DRB3 CCCCAG 4,055 (cox:DRB3) HLA-DRB4 DRB1-short-R10223 CTCTGTGCAGATTCRGACC 2 4,543 (mann:DRB4) hLA-DRB5 GRGC 4,052 (DRB5)HLA-DQB1 DQB1-short-F1.1 26 TGTAAAATCAGCCCGACTG 3 3,915 CCTCTTCDQB1-short-F1.2 25 GCAAAATCAACCCGACTGC 4 CTCTTC DQB1-short-R1 24GGGCAGATTCAGAYTGAGC 5 CCCTA HLA-DPB1 DPB1-short-F1 21TGCTCGCCCCTCCCTAGTG 6 5,405 AT DPB1-short-R1 29 TCAATGTCTTACTCYGGGC 7AGAATCAGAC

Also, in the present invention, a set of primers for PCR of a regionincluding exon 2 and exon 3 known to be highly polymorphic in each ofgenes of HLA-A, HLA-B and HLA-C, which are classic class I antigens, canbe used in combination with the set of primers specific for the classicclass II antigens. The set of primers specific for each of HLA-A, HLA-Band HLA-C genes can be exemplified as follows.

In Table 2, SEQ ID NOs: 8 to 10 represent a set of PCR primersspecifically amplifying an HLA-A gene, which is an α chain of MHC classI. These primers of the set are nucleotide sequences located atpositions, which correspond to the upstream and downstream of allregions of an HLA-A gene (including promoter, exons and introns) andsandwich the all regions, in a human genome sequence (Referencesequence: hg19).

SEQ ID NO: 8 or 9 has a nucleotide sequence corresponding to the29909483rd position to the 29909514th position in a human genomesequence (Reference sequence: hg19).

SEQ ID NO: 10 has a complementary nucleotide sequence to a nucleotidesequence corresponding to the 29914925th position to the 29914954thposition in a human genome sequence (Reference sequence: hg19).

The length of a PCR product obtained by using this primer set isestimated from the reference sequence as about 5,500 bases (bp).

In Table 2, SEQ ID NOs: 11 and 12 represent a set of PCR primersspecifically amplifying an HLA-B gene, which is an α chain of MHC classI. These primers of the set are nucleotide sequences located atpositions, which correspond to the upstream and downstream of allregions of an HLA-B gene (including promoter, exons and introns) andsandwich the all regions, in a human genome sequence (Referencesequence: hg19).

SEQ ID NO: 11 has a complementary nucleotide sequence to a nucleotidesequence corresponding to the 31325796th position to the 31325824thposition in a human genome sequence (Reference sequence: hg19).

SEQ ID NO: 12 has a nucleotide sequence corresponding to the 31321210thposition to the 31321235th position in a human genome sequence(Reference sequence: hg19).

The length of a PCR product obtained by using this primer set isestimated from the reference sequence as about 4,600 bases (bp).

In Table 2, SEQ ID NOs: 13 to 15 represent a set of PCR primersspecifically amplifying an HLA-C gene, which is an α chain of MHC classI. These primers of the set are nucleotide sequences located atpositions, which correspond to the upstream and downstream of allregions of an HLA-C gene (including promoter, exons and introns) andsandwich the all regions, in a human genome sequence (Referencesequence: hg19).

SEQ ID NO: 13 or 14 has a complementary nucleotide sequence to anucleotide sequence corresponding to the 31240868th position to the31240896th position in a human genome sequence (Reference sequence:hg19).

SEQ ID NO: 15 has a nucleotide sequence corresponding to the 31236075thposition to the 31236114th position in a human genome sequence(Reference sequence: hg19).

The length of a PCR product obtained by using this primer set isestimated from the reference sequence as about 4,800 bases (bp).

TABLE 2 Length Estimated length of HLA of SEQ PCR product based class IName of primer Primer sequence ID on the reference gene primer (mer)(5′-3′) NO sequence (hg19)(bp) HLA-A A_F1.4 32 CAGAAACTCAGAGCTAAGG  85,472 AATGATGGTAAAT A_F2.4 32 CAGAAACTCAGAGCTATGG  9 AATGATGGTAAATA_R1.2 30 GCATATAACCATCATCGTG 10 TCCCAAGGTTC HLA-B B_F1.4 29GGTTCCGGTTGCAATAGA 11 4,615 CAGTAACAAA B_R1.2 26 ACGGGTCCAATTTCACAGA 12CAAATGT HLA-C C_F1.4 29 ACACTGCTTAGATGTGCAT 13 4,822 AGTTCACGAA C_F2.429 ACACTGCTTAGATGTGCAT 14 AGTTCCGGAA C_R1.16 40 GAACAATTCTAGACTATGG 15ACCCAATTTTACAAACAAA TA

One or two or more sets of primers described in the presentspecification may be used in a single container for PCR amplifying eachHLA gene by, for example, a multiplex PCR method.

(2) Step of PCR Amplification

In the method of the present invention, a test sample (DNA) is amplifiedby a PCR method using the set of primers prepared in the above step (1).

The PCR amplification reaction is performed in accordance with a generalprotocol and more specifically, as follows.

1. DNA is extracted from a test sample depending upon the form of thesample.

2. The DNA extracted is quantified and the concentrations of primers areappropriately set to prepare the reaction solution.

3. Reaction conditions are set and a PCR is performed.

For example:

Thermal denaturation step (usually 92 to 98° C.)

Annealing step (usually 55 to 72° C.)

Extension step (usually 65 to 80° C.)

In the method of the present invention, the temperature of the annealingstep and the extension step is set preferably at about 65 to 70° C.,more preferably at 65 to 68° C. Owing to the annealing and extension atabout 65 to 70° C., HLA alleles can be produced at the equivalent ratio(uniformly).

4. The obtained PCR product is purified and subjected to the followingnucleotide sequencing step.

The enzyme (DNA polymerase) used in the present invention is notparticularly limited and may be commercial products. Examples caninclude PrimeSTAR® GXL DNA Polymerase, Tks Gflex® DNA Polymerase andTaKaRa LA Taq® (manufactured by TaKaRa Bio Inc.).

(3) Step of Nucleotide Sequencing

Next, the nucleotide sequence of the PCR product (amplified DNA)produced in the above step (2) is determined. The step is preferablyperformed by a technique called high throughput sequencing (or ultrahighsequencing, a massive parallel sequencing). With respect to the highthroughput sequencing, see, for example, “Experimental Medicine”, Vol.27, No. 1, 2009 (Yodo-sha).

A high throughput massive parallel sequencer includes a 454 GS system ofRoche, a genome sequencer Ion Torrent PGM™ system by Life TechnologiesCorporation and MiSeq system by illumina, Inc., etc. In the presentspecification, a sequencing method which is employed in a 454 GS systemof Roche will be described below.

1. The PCR product obtained in the above step (2) is broken up by anebulizer into fragments of about 500 bases.

2. To an end of each of the DNA fragments, a DNA adaptor is attached.

3. DNA fragments attached with a DNA adaptor are dissociated into singlestranded DNA fragments, which are allowed to bind to beads via theadaptor. The obtained beads are encompassed and taken in a water-in-oilemulsion. As a result, a micro-reactor environment containing a singleDNA fragment bound to a single bead is formed.

4. Emulsion PCR is performed to amplify each DNA fragment. By thisemulsion PCR, each DNA fragment is clonally amplified in each microreactor. In this manner, many fragments can be simultaneously and inparallel amplified without competition with other sequences.Subsequently, the emulsion is destroyed and beads bound to amplified DNAfragments are collected.

5. The beads are concentrated and loaded in a pico-titer plate. A singlewell of the pico-titer plate has a size enough to place a single bead.

6. Four types of nucleic acids (A, C, G and T) are added in apredetermined order to each bead. Pyrophosphoric acid produced duringincorporation of each added nucleic acid into the DNA sequence via apolymerase is detected with respect to each bead by a fluorescentreaction of luciferase. Based on the intensity of signal and positionaldata in combination, the nucleotide sequence is determined.

(4) Step of DNA Typing

Subsequently, after the sff file obtained in the above step (3) isclassified depending on MID tags, it is compared with data of known HLAalleles within the nucleotide sequencing database. In this manner, theallele type (6 digits or 8 digits levels) contained in the test sampleis determined at the field 3 level or the field 4 level.

In the method of the present invention, typical sets of primers arelisted in Table 1 (described above). The present invention ischaracterized in that primers are designed so as to correspond to theupstream and downstream of exon 2 and exon 3 of each of genes of HLAclass II and sandwich these exons and the sequence of the DNA amplifiedcorresponding to almost all regions is determined. In this manner, phaseambiguity (uncertainty) is eliminated and information on a null allelecan be obtained.

According to the present invention, since sets of primers for HLA genesare designed so as to anneal at the same temperature during PCR, PCRamplification can be simultaneously performed for a plurality of genesin a single PCR apparatus.

Additionally, owing to the primer sets according to the presentinvention, a multiplex PCR wherein a plurality of HLA genes aresimultaneously PCR amplified in one or two tubes can be performed.

Furthermore, it has been confirmed that sets of primers and enzymes usedin the present invention can be applied to a high-speed PCR apparatus.Thus, PCR can be performed more rapidly and accurately than before.

EXAMPLES

The present invention will be more specifically described by way ofExamples below; however, the present invention is not limited to theseExamples.

Example 1

[Purpose]

The purpose of this example is to check the amplification states foreach gene of HLA class II.

[Method]

Using PrimeSTAR® GXL DNA Polymerase (TaKaRa Bio Inc.) as an enzyme,genomic DNA already extracted as a template and primer sets specific toindividual HLA class II genes (see Table 1: SEQ ID NOs: 1 to 7), a PCRwas carried out. The procedure is more specifically as follows.

(1) To 25 ng of a genomic DNA solution, 4 μL of 5× PrimeSTAR® GXLbuffer, 1.6 μL of a dNTP solution, 1 μL of PCR primers (4 pmol/μL) foreach and 0.8 μL of PrimeSTAR® GXL polymerase were added. The wholeamount of the reaction solution was adjusted to be 20 μL with sterilizedwater.

(2) After kept at 94° C. for 2 minutes, the preparation of (1) wassubjected to a step consisting of a reaction at 98° C. for 10 secondsand a reaction at 70° C. for 3 minutes. This step was repeated 30 timesfor PCR. Note that, for the PCR amplification, GeneAmp® PCR System 9700(Life Technologies Corporation) was used. After the PCR, theamplification states of PCR products were checked by an agarose gelelectrophoresis method.

[Results and Discussion]

PrimeSTAR® GXL DNA Polymerase was able to PCR amplify HLA genes. Theresults of performing agarose gel electrophoresis using the PCRamplified products are shown in FIG. 6. It was found that lane 1 was PCRproducts corresponding to regions including exon 2 and exon 3 ofHLA-DRB1, HLA-DRB4 and HLA-DRB5 genes, lane 2 was a PCR productcorresponding to a region including exon 2 and exon 3 of an HLA-DQB1gene, and lane 3 was a PCR product corresponding to a region includingexon 2 and exon 3 of an HLA-DPB1 gene. In FIG. 6, lane M represents aDNA size marker. A single PCR amplified product having a desiredmolecular weight was successfully obtained for each of HLA class IIgenes by using these primers.

Example 2

[Purpose]

The purpose of this example is to determine the potentiality of amultiplex PCR method of 6 loci of HLA genes including HLA-A, HLA-B andHLA-C genes in addition to HLA class II of Example 1.

[Method]

1. Using PrimeSTAR® GXL DNA Polymerase (TaKaRa Bio Inc.), genomic DNAalready extracted from six specimens (Samples 1 to 6 in Table 3) as atemplate and primer sets specific to individual HLA class I and HLAclass II genes (see Table 2: SEQ ID NOs: 8 to 15 and Table 1: SEQ IDNOs: 1 to 7), a PCR was carried out. Note that, the HLA type for each ofthe six specimens has been already revealed and the specimens include acombination of alleles, in which phase ambiguity was observed in aconventional DNA typing method. The procedure is more specifically asfollows.

(1) The PCR was carried out in two 0.2 ml tubes. In short, HLA-A, HLA-Band HLA-C genes were amplified in one of the tubes and HLA-DRB1,HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1 and HLA-DPB1 genes were amplifiedin the other tube.

(2) To 25 ng of a genomic DNA solution, 4 μL of 5× PrimeSTAR® GXLbuffer, 1.6 μL of a dNTP solution, 3.2 to 5 μL of PCR primers (10pmol/μL) for each and 0.8 μL of PrimeSTAR® GXL polymerase were added.The whole amount of the reaction solution was adjusted to be 20 μL withsterilized water.

(3) After kept at 94° C. for 2 minutes, the preparation of (2) wassubjected to a step consisting of a reaction at 98° C. for 10 secondsand a reaction at 70° C. for 3 minutes. This step was repeated 30 timesfor PCR amplification. Note that, for the PCR amplification, GeneAmp®PCR System 9700 (Life Technologies Corporation) was used. After the PCR,the amplification states of PCR products were checked by an agarose gelelectrophoresis method.

2. The nucleotide sequences of the PCR products were determinedspecifically as follows.

(1) A PCR product was purified by MPure XP Kit (Beckman Coulter, Inc.)in accordance with the standard protocol.

(2) The concentration of the purified PCR product was measured byPicoGreen® dsDNA Quantitation Kit (Invitrogen Corp.) in accordance withthe standard protocol.

(3) The purified PCR products derived from class I genes and thepurified PCR products derived from class II genes were mixed in equalamounts.

(4) A solution of the purified PCR products, a concentration of whichwas adjusted to be 500 ng/100 μL, was subjected to construction of arapid library, and then, emulsion PCR and sequencing by GS Junior(Roche) were carried out in accordance with the standard protocol toobtain nucleotide sequences of 15,000 reads per sample.

(5) A search for homology between these nucleotide sequences and knownnucleotide sequences of HLA alleles on an IMGT HLA database wasperformed to select candidate alleles.

(6) The sequences of the candidate alleles were used as a reference.Mapping was performed by GS Reference Mapper (Roche) on condition thatthe reference matches the read perfectly. The mapping state was checkedvisually to identify an HLA allele.

[Results and Discussion]

1. The results of performing agarose gel electrophoresis using the PCRamplified products are shown in FIG. 7. In FIG. 7, lanes 1 to 6correspond to PCR products obtained using Sample ID 1 to Sample ID 6 ofTable 3. Lane M represents a DNA size marker. As is evident from FIG. 7,a PCR product and a single PCR amplified product having a desiredmolecular weight were successfully obtained for each of HLA class I andHLA class II genes in all of the samples by using the primers shown inTables 1 and 2. Furthermore, the nucleotide sequences of the PCRproducts were determined by the Sanger method. As a result, HLA alleleswere obtained in consistent with known documents. Accordingly, DNAtyping of HLA genes can be performed by the PCR system using the primersshown in Tables 1 and 2.

2. Using six specimens containing a combination of alleles, in whichphase ambiguity is observed in a conventional DNA typing method, a PCRwas performed. PCR products derived from the regions including exon 2and exon 3 of HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4,HLA-DRB5, HLA-DQB1 and HLA-DPB1 genes were subjected to DNA typing ofHLA genes by GS Junior. As a result, DNA typing of all of the genes wassuccessfully made at a 6-digit level (Table 3). From this, the method ofthe present invention can perform DNA typing of HLA genes at a 6-digitor higher level without phase ambiguity and can efficiently detect asubstitution, an insertion and a deletion of bases even in introns,which may be causes of a null allele.

TABLE 3 Sample Allele 1 Allele 2 ID read read radio 1 A*26 01.01 961A*31:01:02 978 0.98 B*15:01:01:01 996 B*35:01:01:02 966 1.03C*03:04:01:02 613 C*07:02:01:04 669 0.92 DRB1*09:01:02:(01) 1526DRB1*13:02:01:(02) 1929 0.79 DRB3*03:01:01:(01) 1675 DRB4*01:03:02:(01)1622 DQB1*03:03:02:02 331 DQB1*06:04:01:(01) 244 1.36 DPB1*02:01:02 117DPB1*04:01:01 120 0.98 2 A*02:03:01 1466 A*24:02:01:01 1419 1.03B*38:02:01 1134 B*54:01:01 1282 0.88 C*01:02:01 1349 C*07:02:01:05 14210.95 DRB1*04:03:01:02 2543 DRB1*08:03:02:02 3773 0.67 DRB4*01:03:01:(06)3664 — DQB1*03:02:01 595 DQB1*06:01:01 536 1.11 DPB1*13:01 484DPB1*19:01 459 1.05 3 A*24:02:01:01 936 A*33:03:01 948 0.99 B*44:03:011219 B*48:01:01 1165 1.05 C*08:03:01 1161 C*14:03 1153 1.01DRB1*13:02:01:(02) 1989 DRB1*16:02:01:(02) 1593 1.25 DRB3*03:01:01:(01)1829 DRB5*02:02:(01):(01) 584 DQB1*05:02:01(01) 316 DQB1*06:04:01:(01)354 0.89 DPB1*04:01:01 119 DPB1*05:01:01 115 1.03 4 A*02:06:01 1178A*24:02:01:01 1138 1.04 B*52:01:01:02 1278 B*54:01:01 1415 0.90C*01:02:01 1277 C*12:02:02 1320 0.97 DRB1*04:05:01:(01) 2001DRB1*15:02:01 2290 0.87 DRB4*01:03:01:(04) 3175 DRB5*01:02:(01):(01) 476DQB1*04:01:01:(01) 468 DQB1*06:01:01 365 1.28 DPB1*05:01/135:01 316DPB1*09:01 304 1.04 5 A*24:02:01:01 1120 A*33:03:01 1180 0.95 B*40:02:01448 B*58:01:01 472 0.95 C*03:02:02:01 780 C*03:04:01:02 781 1.00DRB1*03:01:01:01/02 4071 DMB1*08:02:01:(01) 4288 0.95DRB3*02:02:(01):(01) 2563 — DQB1*02:01:01 175 DQB1*04:02:01:(01) 4330.40 DPB1*02:01:02 194 DPB1*05:01/135:01 236 0.82 6 A*03:02:01 2023A*24:02:01:01 1679 1.20 B*07:02:01 1281 B*13:02:01 1226 1.04C*06:02:01:01 907 C*07:02:01:03 1028 0.88 DRB1*01:01:01 2022DRB1*07:01:01:01 2461 0.82 DRB4*01:03:01:01/03 2158 — DQB1*02:02:01 221DQB1*05:01:01:(03) 453 0.49 DPB1*05:01/135:01 301 DPB1*09:01 300 1.00*radio = allele1/allele2 *DPB1 shows the number of reads of only exon 2and exon 3.

Example 3

[Purpose]

The purpose of this example is to determine the potentiality of amultiplex PCR method of 7 loci of HLA genes (HLA-A, HLA-B, HLA-C,HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 genes) and 9 loci of HLA genes(HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQB1and HLA-DPB1 genes).

[Method]

1. Using PrimeSTAR® GXL DNA Polymerase (TaKaRa Bio Inc.), genomic DNAalready extracted from four specimens (Samples 1 to 4 in Table 4) as atemplate and primer sets specific to individual HLA class I and HLAclass II genes (see Table 2: SEQ ID NOs: 8 to 15 and Table 1: SEQ IDNOs: 1 to 5), a PCR was carried out. Note that, the HLA type for each ofthe four specimens has been already revealed and the specimens include acombination of alleles, in which phase ambiguity was observed in aconventional DNA typing method.

SEQ ID NOs: 8 to 15 of Table 2 and SEQ ID NOs: 1 to 5 of Table 1 wereused with respect to HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4,HLA-DRB5 and HLA-DQB1 genes. DPB1-F2 (5′-CTCAGTGCTCGCCCCTCCCTAGTGAT-3′:SEQ ID NO: 16) and DPB1-R2 (5′-GCACAGTAGCTTTCGGGAATTGACCA-3′: SEQ ID NO:17) were used with respect to an HLA-DPB1 gene. DPB1-F2 (SEQ ID NO: 16)and DPB1-R2 (SEQ ID NO: 17) are represent a set of PCR primersspecifically amplifying an HLA-DPB1 gene, which is a β chain of MHCclass II. These primers of the set are nucleotide sequences located atpositions, which correspond to the upstream and downstream of exon 2 toa 3′ untranslated region of an HLA-DPB1 gene and sandwich the region, ina human genome sequence (Reference sequence: hg19). SEQ ID NO: 16 has anucleotide sequence corresponding to the 33048182nd position to the33048207th position in a human genome sequence (Reference sequence:hg19). SEQ ID NO: 17 has a complementary nucleotide sequence to anucleotide sequence corresponding to the 33055428th position to the33055453rd position in a human genome sequence (Reference sequence:hg19). The length of a PCR product obtained by using this primer set isestimated from the reference sequence as about 7,300 bases (bp).

The procedure is more specifically as follows.

(1) The PCR was carried out in two 0.2 ml tubes. In short, HLA-A, HLA-B,HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 genes were amplified inone of the tubes. HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DRB3, HLA-DRB4,HLA-DRB5, HLA-DQB1 and HLA-DPB1 genes were amplified in the other tube.

(2) To 25 ng of a genomic DNA solution, 4 μL of 5× PrimeSTAR® GXLbuffer, 1.6 μL of a dNTP solution, 3.2 to 5 μL of PCR primers (10pmol/μL) for each and 0.8 μL of PrimeSTAR® GXL polymerase were added.The whole amount of the reaction solution was adjusted to be 20 μL withsterilized water.

(3) After kept at 94° C. for 2 minutes, the preparation of (2) wassubjected to a step consisting of a reaction at 98° C. for 10 secondsand a reaction at 70° C. for 3 minutes. This step was repeated 30 timesfor PCR amplification. Note that, for the PCR amplification, GeneAmp®PCR System 9700 (Life Technologies Corporation) was used. After the PCR,the amplification states of PCR products were checked by an agarose gelelectrophoresis method.

2. The nucleotide sequences of the PCR products were determinedspecifically as follows.

(1) A PCR product was purified by AMPure XP Kit (Beckman Coulter, Inc.)in accordance with the standard protocol.

(2) The concentration of the purified PCR product was measured byPicoGreen® dsDNA Quantitation Kit (Invitrogen Corp.) in accordance withthe standard protocol.

(3) The purified PCR products derived from class I genes and thepurified PCR products derived from class II genes were mixed in equalamounts.

(4) A solution of the purified PCR products, a concentration of whichwas adjusted to be 500 ng/100 μL, was subjected to construction of alibrary, and then, emulsion PCR and sequencing by Ion PGM (LifeTechnologies Corporation) were carried out in accordance with thestandard protocol to obtain nucleotide sequences of 400,000 reads persample.

(5) A search for homology between these nucleotide sequences and knownnucleotide sequences of HLA alleles on an IMGT HLA database wasperformed to select candidate alleles.

(6) The sequences of the candidate alleles were used as a reference.Mapping was performed by GS Reference Mapper (Roche) on condition thatthe reference matches the read perfectly. The mapping state was checkedvisually to identify an HLA allele.

[Results and Discussion]

1. The results of performing agarose gel electrophoresis using the PCRamplified products are shown in FIG. 8. In FIG. 8, lanes 1 to 4correspond to PCR products obtained using Sample ID 1 to Sample ID 4 ofTable 4. The leftmost lane represents a DNA size marker. As is evidentfrom FIG. 8, a PCR product and a single PCR amplified product having adesired molecular weight were successfully obtained for each of genes inall of the samples of both 7 loci of HLA genes and 9 loci of HLA genesby using the primers described above.

2. Using four specimens containing a combination of alleles, in whichphase ambiguity is observed in a conventional DNA typing method, a PCRwas performed. PCR products derived from each of the genes weresubjected to HLA typing by Ion PGM. As a result, DNA typing of all ofthe genes was successfully made at a 6-digit level (Table 4). From this,the method of the present invention can perform DNA typing of HLA genesat a 6-digit or higher level without phase ambiguity and can efficientlydetect a substitution, an insertion and a deletion of bases even inintrons, which may be causes of a null allele.

The material in the ASCII text file, named “WING2-56186-SeqLst.txt”,created May 24, 2016, file size of 4,096 bytes, is hereby incorporatedby reference.

TABLE 4 sample ID 1 sample ID 2 sample ID 3 sample ID 4 Allele 1 Allele2 Allele 1 Allele 2 Allele 1 Allele 2 Allele 1 Allele 2 Multiplex methodof 7 loci of HLA genes A*03:01:01 A*31:01:02 A*02:05:01 A*03:01:01A*11:01:01 A*32:01:01 A*11:01:01 A*23:01:01 B*07:02:01 B*40:01:02B*47:01:01 B*50:01:01 B*07:02:01 B*51:01:01 B*35:01:01 B*49:01:01C*03:04:01 C*07:02:01 C*06:02:01 — C*07:02:01 C*15:02:01 C*04:01:01C*07:01:01 DRB1*04:04:01 DRB1*15:01:01 DRB1*07:01:01 — DRB1*15:01:01 —DRB1*04:01:01 DRB1*10:01:01 DRB4*01:03:01 DRB4*01:01:01 DRB5*01:01:01DRB4*01:03:01 DRB5*01:01:01 Multiples method of 9 loci of HLA genesA*03:01:01 A*31:01:02 A*02:05:01 A*03:01:01 A*11:01:01 A*32:01:01A*11:01:01 A*23:01:01 B*07:02:01 B*40:01:02 B*47:01:01 B*50:01:01B*07:02:01 B*51:01:01 B*35:01:01 B*49:01:01 C*03:04:01 C*07:02:01C*06:02:01 — C*07:02:01 C*15:02:01 C*04:01:01 C*07:01:01 DRB1*04:04:01DRB1*15:01:01 DRB1*07:01:01 — DRB1*15:01:01 — DRB1*04:01:01DRB1*10:01:01 DRB4*01:03:01 DRB4*01:01:01 DRB5*01:01:01 DRB4*01:03:01DRB5*01:01:01 DQB1*03:02:01 DQB1*06:02:01 DQB1*02:02:01 — DQB1*06:02:01— DQB1*03:02:01 DQB1*05:01:01 DPB1*04:01:01 — DPB1*04:01:01 DPB1*15:01DPB1*04:01:01 DPB1*10:01 DPB1*02:01:02 DPB1*04:01:01

The invention claimed is:
 1. A method for DNA typing of a plurality ofHLA genes from a human genomic sequence at a six-digit or higher level,comprising the following steps: (1) a step of preparing a set of primersfor at least three target genes from the human genome sequencecomprising (1) a first target gene comprising one or more of HLA-DRB1,HLA-DRB3, HLA-DRB4, or HLA-DRB5, (ii) a second target gene comprisingHLA-DQB1, and (iii) a third target gene comprising HLA-DPB1, wherein theprimers anneal specifically to an intron 1 and an exon 4 region,respectively, of each of the three target genes, and amplify a regioncomprising exon 2, intron 2, exon 3, intron 3, and a part of exon 4 ofeach of the three target genes; wherein the set of primers comprisesoligonucleotides having nucleotide sequences as shown in SEQ ID NOs: 1and 2, respectively, for amplifying the region of the first target gene;wherein the set of primers comprises oligonucleotides having nucleotidesequences as shown in any one of SEQ ID NOs: 3 or 4, or both, and SEQ IDNO: 5, respectively, for amplifying the region of the second targetgene; and wherein the set of primers comprises oligonucleotides havingnucleotide sequences as shown in SEQ ID NOs: 6 and 7, respectively, foramplifying the region of the third target gene; (2) a step of PCRamplifying a test sample (DNA) using the set of primers in a multiplexPCR method to obtain PCR amplified products of each of the at leastthree target genes; (3) a step of determining the nucleotide sequencesof the PCR amplified products; and (4) optionally, a step of carryingout a homology search within a database.